The Na channel is an integral membrane protein central to signaling in the heart and other excitable tissues. The pores of ion channels are principal determinants of ion selectivity, drug binding and gating. The determination of the crystal structure of an inwardly rectifying K channel was an important advance that provided a framework for testing hypotheses concerning the pore structure of related channels. Nevertheless, as such crystal structures have the important limitation that movement, a feature of the pore that is vitally important to channel function, is imperceptible. Thus, this proposal will emphasizes vital approaches to structure-function analysis of the permeation pathway with an emphasis on understanding the role of the pore in fundamental mechanisms of channel gating. This proposal builds on the work from the prior period of support and will test the hypotheses that: 1. Motion in the outerpore mouth underlies slow forms of inactivation of the channel. We will use the complementary approaches of measuring the state-dependence of spontaneous and induced disulfide bond formation and fluorescence resonance energy transfer (FRET) in channels with paired cysteine substitutions in the outer pore. 2. The structure of the outer pore can be further refined by studying the blocking characteristics of mu-conotoxins. Paired mutations in the channel protein and toxin and the analytic techniques of mutant cycle analysis and electrostatic compliance will be used to estimate molecular distances between toxin and channel residues. 3. The cytoplasmic portions of the S6 segments of the channels form a part of the activation gate. Cysteine mutations in the S6 segments of all channel domains will be expressed on an inactivation-deficient channel background to determine the state-dependent accessibility to thiol-specific modifying reagents, block by the group II metal ions, Cd 2+ and Zn 2+ and state dependent block by quarternary ammonium derivatives of local anesthetic antiarrhythmic drugs. 4. Calcium/calmodulin signaling regulates Na channel gating in a physiologically significant, isoform-specific manner. Using electrophysiological, biochemical and fluorescence measurements we will test the hypothesis that CaM is constitutively tethered to the channel has direct and indirect (through CaCaMKinase) effects on Na channel isoforms that modulate inactivation gating. Given the central role of the Na channel in normal physiology and disease (arrhythmias, myotonia and epilepsy) this proposal promises to further our understanding of permeation and gating, pathophysiological mechanisms of diseases of excitability and the mechanism of action of clinically useful drugs (antiarrhythmic percent local anesthetic percent and anticonvulsants). [unreadable] [unreadable]